Received on January 22, 2009
Accepted on May 26, 2009

Molecular Diagnostics and Genetics

CYP2D6 Genotyping for Functional-Gene Dosage Analysis by Allele Copy Number Detection

¹ Laboratory for Genotyping Development, Human Genome Center, The Institute of Medical Science, University of Tokyo, Tokyo, Japan
² Laboratory for Medical Informatics, Human Genome Center, The Institute of Medical Science, University of Tokyo, Tokyo, Japan
³ Laboratory for Pharmacogenetics, Center for Genomic Medicine, RIKEN, Yokohama, Japan
⁴ Department of Laboratory of Molecular Medicine, Human Genome Center, The Institute of Medical Science, University of Tokyo, Tokyo, Japan
⁵ Laboratory for Genotyping Development, and Laboratory of Molecular Medicine, Human Genome Center, The Institute of Medical Science, University of Tokyo, Tokyo, Japan

^* To whom correspondence should be addressed. E-mail: nhosono{at}src.riken.jp.

BACKGROUND: Cytochrome P450 2D6 (CYP2D6), one of the most important drug-metabolizing enzymes, has been reported to possess variation in the encoding CYP2D6 gene (cytochrome P450, family 2, subfamily D, polypeptide 6) that affects enzymatic activity. For the pharmacogenetic study of CYP2D6, accurate measurement of the dosage of the functional gene is essential; however, current genotyping techniques are insufficient because of their inability to provide the exact copy number of functional CYP2D6 genes.

METHODS: We developed 3 quantitative real-time PCR (qPCR) assays for estimating the total copy number of the CYP2D6 gene, as well as 24-multiplex PCR-based real-time Invader assays (mPCR-RETINAs) for estimating the allele ratio at each variation locus. After determining the allele copy number at each locus, we estimated the frequencies of CYP2D6 alleles in a population and the diplotype in each individual by a copy number variation phaser. The qPCR assays and RETINAs used for HapMap Japanese and Chinese samples were applied to 455 Japanese individuals.

RESULTS: Forty-two individuals (9.2%) had one CYP2D6 gene copy, 207 (45.5%) had 2 copies, 161 (35.4%) had 3 copies, 40 (8.8%) had 4 copies, and 5 (1.1%) had 5 copies of the CYP2D6 gene. We found 16 different CYP2D6 alleles, with frequencies similar to those described in previous reports. In the diplotype analysis, we observed that CYP2D6*1/*1 and *1/*10-*36 were the most common diplotypes (approximately 20%) in our population.

CONCLUSION: Our method is the first to determine the exact number of functional CYP2D6 gene copies. We believe our method will facilitate and accelerate the detailed pharmacogenetic analysis of CYP2D6.